(1) Field of the Invention
This invention relates to a method for purifying single-chain and/or double-chain tissue plasminogen activator (hereinafter referred to as sc-TPA and dc-TPA, respectively) from a mixture containing sc-TPA and/or dc-TPA.
(2) Description of the Prior Art
tPA (tissue plasminogen activator) is a protein which is produced in a tissue of a higher animal, serves to activate plasminogen, a precursor of plasmin which is known to be a proteolytic enzyme specifically to fibrin, and has now being brought into a focus as a potential thrombolytic agent.
tPA has two molecular forms, single-chain tPA and double-chain tPA, having the same molecular weight (about 70,000 daltons) and is obtained as a mixture in ordinary procedures for tPA production. A double-chain form has about 10-fold higher activity for activation of plasminogen than a single-chain form (European Patent Publication No. 112122A). However, it is known that sc-TPA has a stronger capacity of binding to fibrin than dc-TPA and, once bound to fibrin, quickly converted to dc-TPA (D. C. Rijken et al., J. Biol. Chem. 257, 2920-2925, 1982). Consequently, in order to realize an effective treatment of thrombosis by tPA, an increased binding capacity of tPA to fibrin in the clotting of blood must be obtained by using a tPA mixture containing an increased amount of sc-TPA or by using a preparation containing sc-TPA only.
Under these circumstances and also in order to investigate properties and functions of tPA in more detail, there has been a great need for improving methods for obtaining single-chain form exclusively and for isolating single-chain and double-chain form separately from a mixture thereof.
A method previously known for preparing tPA, for example, comprises culturing cells, which are indigenously capable of producing tPA or manipulated to carry the tPA gene, and then isolating tPA from the resultant cultured cells and/or from the culture supernatant. For example, the above-mentioned European Patent Publication No. 112122A disclosed a method of purifying tPA using an Erythrina trypsin inhibitor (hereinafter referred to as ETI), or an immobilized Kunits inhibitor, which is produced in seeds of Erythrina latissima and other Erythrina plants and acts as an inhibitor to trypsin, plasmin and tPA but not to urokinase. In this method, however, a separate isolation of sc-TPA from dc-TPA was not taken into consideration.
In a method previously known for preparing sc-TPA, a proteinase inhibitor such as Aprotinin is added to a culture medium to suppress conversion of tPA from single-chain to double-chain form (D. C. Rijken et al., J. Biol. Chem. 256, 7035-7041, 1981).
In another method for purifying sc-TPA, an immobilized monoclonal antibody which adsorbs specifically sc-TPA is used (catalogue by BioPool, Sweden). This method, however, is inferior to the method with ETI in respect to the capacity of adsorbing tPA and stability of a column to be used. Moreover and disadvantageously, this method can not remove an impurity which is a protein with a molecular weight of 110,000.+-.20,000 daltons and potentially acts as an antigen to react with an anti-human tPA antibody.